Field of the Invention
The invention relates to the measurement of activity of protein inhibitors.
Summary of the Related Art
Potent and selective inhibitors of enzymes and other bioactive proteins are used for numerous medical and biotechnological applications, as well as for research. The potency of a protein inhibitor is usually measured in cell-free assays, using purified protein preparations. The selectivity of the inhibitor is typically deduced by comparing its activities for the inhibition of different proteins. It is well known in the art, however, that the inhibitory activity of a compound, which is usually expressed as IC50 (the concentration required for half-maximal protein activity inhibition) or Ki (the measure of bond-tightness between the enzyme and its inhibitor) may vary depending on many factors. In the case of enzyme inhibitors, these factors may include (i) the nature of enzyme preparation (for example, what type of cells the recombinant protein was expressed in), (ii) the quality of the enzyme preparation, (iii) the nature and the quality of additional subunits of a multi-subunit enzyme, (iv) the nature and quality of the substrate used in the enzymatic reaction, (v) the buffer composition and temperature conditions of the reaction, and other variable factors.
As a recent example, Echalier et al. (Chemistry & Biology 19, 1028-1040, 2012) show that Ki values for cyclin-dependent kinase (CDK) inhibitors, were different when the CDKs were combined with different regulatory cyclin proteins. On the other hand, U.S. Provisional Application No. 61/734,127 teaches that both the Km values for ATP (used in the assays for IC50 determination) and the relative activity of a CDK inhibitor, Purvalanol A, for CDK2 and CDK3 kinases were different from the values reported by Echalier et al., in the assays conducted using different enzyme preparations, phosphorylation substrates and ATP concentrations.
The utility of a protein inhibitor lies in its effect on the target protein within a cell rather than in an artificial cell-free assay. Therefore the most pertinent inhibitory activity is the one measured in a cell-based assay, which measures directly the effect of the inhibitor on the activity of the target protein in an intact cell. However, direct cell-based assays are available for only a small fraction of protein targets. The most widely used class of such assays is aimed at cell surface receptors of specific ligands, where the activity of a receptor's antagonist is measured by the competition for receptor-specific ligand binding at the cell surface. Another general class of cell-based assays is based on the use of promoter-reporter constructs where the promoter activity is dependent on the binding of a specific transcription factor to its binding site in a promoter. The ability of a compound to inhibit the transcription factor activity within the cell can be measured by its effect on the reporter expression from the promoter (In the latter case, however, the inhibition of promoter activity may also reflect an indirect effect on the transcription factor.)
For most of the intracellular proteins, however, target-specific cell-based assays are not available. For example, a cell-based assay for a specific protein kinase could measure the phosphorylation of an intracellular protein substrate, provided that the substrate is phosphorylated only by the target kinase. However, there are very few, if any, examples of a protein being phosphorylated by only a single kinase. Hence, there is great need in the art for a different type of cell-based assays specific for a particular protein.